Abstract Tuberculosis (TB) represents a severe world health problem. TB accounts for 1.5 million deaths annually and a third of the global population is believed to be infected with Mycobacterium tuberculosis (Mtb), the causative agent of TB. A major challenge to controlling TB is the rise in multidrug and extensively drug resistant (MDR and XDR) Mtb strains. Because of its remarkable sterilizing activity as well as synergistic activity with other drugs, pyrazinamide (PZA) is a critical component of the current four drug regimen for drug sensitive TB and it is also included in many treatment regimens for MDR-TB. However, the future utility of PZA is in jeopardy because of the rise in PZA resistant Mtb strains. PZA is a prodrug that is converted to the active moiety pyrazinoic acid (POA) by the pyrazinamidase PncA, and the most common category of PZA-resistant Mtb strains harbor mutations in the pncA gene. As an alternative to PZA, direct dosing with pyrazinoic acid (POA) has been proposed. In this R21, we propose to evaluate inhaled POA as an adjunctive therapy for TB using a guinea pig model. By directly delivering drug to the lungs, locally high concentrations at the site of Mtb infection but systemically low doses can be achieved. In Aim 1 we will perform pharmacokinetic (PK) studies of dry powder aerosols of POA delivered by inhalation to guinea pigs. In Aim 2 we will perform an efficacy experiment to assess the effect of inhaled POA as an adjunctive therapy for treating TB in guinea pigs. The combined results of Aims 1 and 2 will test our hypotheses that high levels of POA can be achieved in the lungs and airway by inhaled therapy and that treatment with inhaled POA as a supplement to standard therapy will significantly reduce the organ burden of Mtb bacteria and the course of TB disease. Our plan has high clinical significance and potential to reveal inhaled POA as a new adjunctive therapy for treating drug sensitive TB and an approach for rescuing the use of PZA as an important class of TB drug.